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Active cyclin B1–Cdk1 first appears on centrosomes in prophase

Nature Cell Biology volume 5, pages 143–148 (2003)Cite this article

Abstract

Cyclin B1–Cdk1 is the key initiator of mitosis, but when and where activation occurs has not been precisely determined in mammalian cells. Activation may occur in the nucleus or cytoplasm, as just before nuclear envelope breakdown, Polo-like kinase1 (Plk1) is proposed to phosphorylate cyclin B1 in its nuclear export sequence (NES), to trigger rapid nuclear import. We raised phospho-specific antibodies against cyclin B1 that primarily recognise the active form of the complex. We show that cyclin B1 is initially phosphorylated on centrosomes in prophase and that Plk1 phosphorylates cyclin B1, but not in the NES. Furthermore, phosphorylation by Plk1 does not cause cyclin B1 to move into the nucleus. We conclude that cyclin B1–Cdk1 is first activated in the cytoplasm and that centrosomes may function as sites of integration for the proteins that trigger mitosis.

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Figure 1: Specificity of anti-cyclin B1 S126-P and anti-cyclin B1 S133-P antibodies.
Figure 2: S126-P and S133-P forms of cyclin B1 appear first on centrosomes in prophase.
Figure 3: Plk1 preferentially phosphorylates cyclin B1 on Ser 133.
Figure 4: Plk1 phosphorylates S133 in vivo without triggering mitosis.
Figure 5: Plk1 does not trigger the import of cyclin B1 to the nucleus.

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Acknowledgements

We are indebted to C. Broad for help with the cell culture. We are grateful to S. Kornbluth for discussing results before publication, to J. Gannon and T. Hunt for anti-Cdk1 antibodies, and K. Lee and R. Erikson (Havard, USA) for the Plk1 baculovirus. We thank E. Nishida (Kyoto, Japan) for sending us anti-phospho-Ser 147 serum. This work was supported by a Wellcome Trust Training Fellowship to CL, by the Association for International Cancer Research [AICR], and by a programme grant to JP from Cancer Research UK (CRC).

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Author notes

  1. Mark Jackman, Catherine Lindon and Erich A. Nigg: These authors contributed equally to this work

Authors and Affiliations

  1. Wellcome Trust/ Cancer Research UK Institute and Department of Zoology, Tennis Court Road, Cambridge, CB2 1QR, UK

    Mark Jackman, Catherine Lindon & Jonathon Pines

  2. Department of Cell Biology, Max-Planck Institute for Biochemistry, Martinsried, 82152, Germany

    Erich A. Nigg

Authors
  1. Mark Jackman
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  4. Jonathon Pines
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Correspondence to Jonathon Pines.

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Supplementary information

Supplementary Figures

Figure S1 S126-P antibody staining focuses at centrosomes. (PDF 470 kb)

Figure S2 PLK1 preferentially phosphorylates S133 and not S147 in cyclin B1

Figure S3 Plk1 accumulates in the nucleus at prophase before cyclin B1.

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Jackman, M., Lindon, C., Nigg, E. et al. Active cyclin B1–Cdk1 first appears on centrosomes in prophase. Nat Cell Biol 5, 143–148 (2003). https://doi.org/10.1038/ncb918

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